FLAMBE: A kinetic fluorescence polarization assay to study activation of monomeric BAX

Jarvier N. Mohammed, Jesse D. Gelles, Jerry Edward Chipuk

Research output: Contribution to journalArticlepeer-review

2 Scopus citations

Abstract

BAX activation techniques are crucial to studying the intrinsic pathway of apoptosis— thousands of pro-apoptotic signals converge on BAX activation. Current methodologies are predominantly limited to membrane permeabilization studies, which assess endpoint functionality of oligomeric BAX, but overlook early activation steps of cytosolic BAX. Here we detail FLAMBE: a fluorescence polarization ligand assay for monitoring BAX early-activation in solution. We also describe a dual-metric parameterization strategy for distillation of kinetic data and comparative analyses when studying candidate ligands. For complete details on the use and execution of this protocol, please refer to Gelles et al. (2022).

Original languageEnglish
Article number101252
JournalSTAR Protocols
Volume3
Issue number2
DOIs
StatePublished - 17 Jun 2022

Keywords

  • Cell Biology
  • Cell-based Assays
  • Molecular Biology
  • Molecular/Chemical Probes
  • Signal Transduction
  • Single-molecule Assays

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